The src family kinase lck plays a critical role in T lymphocyte development and activation. Expression of lck is regulated by two promoters, termed distal and proximal promoters, in both mice and humans. Although the existence of these promoters was described 20 years ago, their function in developing and mature T cells has not been analyzed. To address this question, we have generated mutant BAC transgenes by deletion of either proximal or distal lck promoter. Studies of these mice have demonstrated selective expression of lck at preferential and different stages of development when driven by one or the other of its two promoters. Corresponding selective stages of T cell development and differentiation have been shown to be dependent upon promoter-specific lck expression. The proximal lck promoter is critical for early stages of thymic T cell development, but insufficient to support later stages of development or mature T cell responses to TCR stimulation. The distal promoter does not support early development, but does support differentiation from DP to mature SP thymocytes, as well as the TCR-mediated response of mature peripheral T cells. Mice expressing proximal and distal promoters on distinct BAC transgenes support wild-type levels of T cell development. The proximal and distal promoters thus function at distinct stages of T cell development and have non-redundant roles in the developmental process. Stimulated by the observation that alternative promoters can have distinct functional roles during development, we have initiated studies in collaboration with the lab of Keji Zhao to analyze genome-wide patterns of differential promoter expression during T cell development. In initial experiments, we have identified multiple genes that are regulated by differential use of alternative promoters during stages of T cell development. Further studies in progress will analyze chromatin structure and epigenetic correlates of gene expression. The developmental stage-specific function of alternative promoters will next be analyzed using CRISPR-Cas methodology already established in our lab. We investigated the function of tumor suppressor p53 in regulating proliferation and function of T lymphocytes. We have made the unexpected observation that antigen-specific proliferative responses of naive and memory CD4 T cells require the down-modulation of tumor suppressor p53. In the absence of TCR signal, IL-2 induces a sustained increase in p53 protein, which prevents proliferative responses despite strong signaling through the IL-2 receptor. In contrast, TCR signaling results in early termination of p53 protein expression by decreasing p53 mRNA as well as by strong transcriptional induction of the p53-regulating protein Mdm2. Down-modulation of p53 in response to antigen stimulation is in fact critical for antigen-specific T cell proliferation; and preventing p53 degradation by inhibiting Mdm2 results in sustained p53 protein levels and prevents antigen-specific T cell proliferation. These studies elucidate a critical role of p53 as a negative regulator of T cell proliferation. It is the termination of p53 elevation by TCR signaling that allows proliferative responses to occur, enforcing antigen specificity. Preliminary studies of p53 effect on antigen-inexperienced and memory T cell repertoire have strongly suggested that p53 affects the threshold of TCR signaling required for activation of unprimed T cells by specific antigen, and their subsequent differentiation into memory T cells. p53 thus appears to be an important regulator of antigen-specific T cell activation and in vivo response, proliferation, and differentiation. T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two distinct antigen-presenting cell populations (APCs), B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent costimulatory pathways. Conventional models describe the expression of both of these costimulatory molecules on the same APC, both for T cell presentation and for cross-regulation of B7 and CD40 expression. Here, we report that, contrary to the conventional paradigm, cellular requirements for B7 and CD40 expression were distinct for GC TFH , GC B cell, and high affinity antibody responses. B7 expression was required on DCs but not on B cells, while CD40 was required on B cells but not DCs; and there was in fact no requirement for co-expression of B7 and CD40 on the same cell for GC responses. Our findings thus identify a much revised model for costimulatory function in the GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by distinct APC populations. T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two distinct antigen-presenting cell populations (APCs)-B cells and dendritic cells (DCs)-in the presence of B7- and CD40-dependent costimulatory pathways. T-APC interactions with both populations are generally considered to depend on similar molecular mechanisms, including the involvement of these two costimulatory receptor-ligand pairs, but direct assessment of the role of each pathway in germinal center-dependent adaptive responses has not been conducted. Here we have utilized models that enable the selective elimination of CD28-B7 and CD40-CD40L signaling during T-DC vs. T-B antigen-driven interactions to probe this issue. In contrast to prevailing views that both pathways are critical for productive T-dependent humoral immunity at both the early (T-DC) and late (T-B) phases of the response, we found that the cellular requirements for B7 and CD40 expression were distinct. B7 expression was required on DCs but not on B cells, while CD40 was required on B cells but not DCs. These data emphasize the emerging evidence that distinct molecular rules apply to CD4+ T cell-myeloid cell and CD4+ T cell-lymphoid cell interactions, with important implications for understanding how to optimize or inhibit these events to promote vaccine responses or limit autoimmunity To further elucidate the role of CD40-CD40L interactions in multiple T cell-developmental and functional events, we are generating unique mouse genetic models. To analyze the role of signaling through CD40 on diverse cell populations, we have produced by CRISPR-Cas9 a set of CD40 cytoplasmic domain mutants that disrupt putative binding sites for TRAFs 2,3, and 6, and have generated initial data indicating differential dependence of these cytoplasmic domains for diverse functions, including B cell germinal center responses, Ig class switching, and affinity maturation; susceptibility to experimental autoimmune encephalomyelitis; in vivo T cell cytokine responses; and iNKT cell selection. To determine CD40L function, we are in the process of generating mutants that express only cell surface or only secreted forms of CD40L.